CRISPR/Cas12a Based Rapid Molecular Detection of Acute Hepatopancreatic Necrosis Disease in Shrimp

• Published in: Frontiers in veterinary science 2022-01, Vol.8, p.819681-819681
• Main Authors: Li, Chenglong, Lin, Nan, Feng, Zhihua, Lin, Minhua, Guan, Biyun, Chen, Kunsen, Liang, Wangwang, Wang, Qiaohuang, Li, Miaomiao, You, Yu, Chen, Qi
• Format: Article
• Language: English
• Subjects: acute hepatopancreatic necrosis disease (AHPND); CRISPR/Cas12a; lateral flow strip (LFS); recombinase polymerase amplification (RPA); shrimp; Veterinary Science
• ISSN: 2297-1769; 2297-1769
• DOI: 10.3389/fvets.2021.819681

Acute hepatopancreatic necrosis disease (AHPND), formerly called early mortality syndrome (EMS), causes high mortality in cultured penaeid shrimp, particularly and . AHPND is mainly caused by species carrying the pVA1 plasmid encoding the virulence genes Photorhabdus insect-related ( ) and . We developed a new molecular assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology (RPA-CRISPR/Cas12a) to detect and , with a fluorescent signal result. The fluorescence RPA-CRISPR/Cas12a assay had a detection limit of 20 copies/μL for and . To improve usability and visualize RPA-CRISPR/Cas12a assay results, a lateral flow strip readout was added. With the lateral flow strip, the RPA-CRISPR/Cas12a assay had a lower limit of detection of 200 copies/μL (0.3 fmol/L). The lateral flow assay can be completed in 2 h and showed no cross-reactivity with pathogens causing other shrimp diseases. In a field test of 60 shrimp samples, the RPA-CRISPR/Cas12a lateral flow assay showed 92.5% positive predictive agreement and 100% negative predictive agreement. As the new RPA-CRISPR/Cas12a assay is rapid, specific, and does not require complicated experimental equipment, it may have important field applications for detecting AHPND in farmed shrimp.